SV40病毒T抗原真核载体的构建与表达  

Construction of a SV40 Virus Large J Antigen Eukaryocyte Vector and Its Targeted Expression

作　　者：王鹰[1] 邓军[1] 杨希川[1] 郝飞[1] 

机构地区：[1]第三军医大学西南医院,重庆400038

出　　处：《中华医院感染学杂志》2006年第3期256-259,共4页Chinese Journal of Nosocomiology

基　　金：国家自然科学基金资助(30371299)

摘　　要：目的设计、构建SV40病毒T抗原真核表达载体,并导入真核细胞进行表达鉴定。方法采用重叠延伸拼接法,从pUC19-SV40载体中亚克隆切除内含子的SV40病毒T抗原基因全长片段,EcoRⅠ/BamHⅠ双酶切后定向克隆至pEGFP-N1真核表达载体上,酶切鉴定重组质粒;用脂质体转染的方法将构建的载体导入原代培养的正常人成纤维细胞进行表达,检测SV40病毒T抗原基因在成纤维细胞内的表达。结果SV40病毒T抗原基因成功克隆至pEGFP-N1真核表达载体中;转染成纤维细胞提取基因组DNA及总RNA,分别经PCR和RT-PCR反应扩增出288 bp的特异性片段。结论本试验构建的SV40病毒T抗原重组质粒为利用SV40T抗原进行真核细胞研究提供了稳定、可靠的分子工具。OBJECTIVE To design and construct eukaryocyte expression vector of SV40 virus large T antigen and induce its targeted expression in eukaryocyte. METHODS SV40 large T gene which excised intron was cloned by SOE （splicing by overlapping extension） and digested with restricted enzymes EcoR I and BamH I . By the same methods, we got the digested product of pEGFP-N1. After that, the two fragments were ligated to form SV40 T-EGFP by Ligation Kit, and sequenced by TaKaRa ABI Prism Terminator Cycle Sequence Kit. The reconstruc- ted vector was transfected into primary cultured human fibroblast using a Lipofectin transfection method. At 48 h after transfection, the expression of SV40T was detected with PCR and RT-PCR using specific primer of T gene. RESULTS The restricted enzymes digested and sequencing results showed that SV40 large T gene had cloned into pEGFP-N1 vector successfully, The genome DNA and total RNA were isolated from the positive cells. With these samples, the specific 288 bp fragment was amplified using PCR and RT-PCR. CONCLUSIONS The recombinant plasmid SV40T.EGFP will be a stable and valuable molecular tool for human eukaryocyte study.

关 键 词：SV40病毒 T抗原 真核载体 表达 

分 类 号：R373[医药卫生—病原生物学]